The synthesis and accumulation of omega-3 long-chain polyunsaturated fatty acids in transgenic Camelina sativa is demonstrated using the so-called alternative pathway. This aerobic pathway is found in a small number of taxonomically unrelated unicellular organisms and utilizes a C18 Δ9-elongase to generate C20 PUFAs. Here, we evaluated four different combinations of seed-specific transgene-derived activities to systematically determine the potential of this pathway to direct the synthesis of eicosapentaenoic acid (EPA) in transgenic plants. The accumulation of EPA and the related omega-3 LC-PUFA eicosatetraenoic acid (ETA) was observed up to 26.4% of total seed fatty acids, of which ETA was 9.5%. Seed oils such as these not only represent an additional source of EPA, but also an entirely new source of the bona fide fish oil ETA. Detailed lipidomic analysis of the alternative pathway in Camelina revealed that the acyl-substrate preferences of the different activities in the pathway can still generate a substrate-dichotomy bottleneck, largely due to inefficient acyl-exchange from phospholipids into the acyl-CoA pool. However, significant levels of EPA and ETA were detected in the triacylglycerols of transgenic seeds, confirming the channelling of these fatty acids into this storage lipid.,© 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.